CRISPR/Cas9-induced (CTG⋅CAG)n repeat instability in the myotonic dystrophy type 1 locus: implications for therapeutic genome editing

Myotonic dystrophy type 1 (DM1) is caused by (CTG⋅CAG)n-repeat expansion within the DMPK gene and thought to be mediated by a toxic RNA gain of function. Current attempts to develop therapy for this disease mainly aim at destroying or blocking abnormal properties of mutant DMPK (CUG)n RNA. Here, we explored a DNA-directed strategy and demonstrate that single clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-cleavage in either its 5′ or 3′ unique flank promotes uncontrollable deletion of large segments from the expanded trinucleotide repeat, rather than formation of short indels usually seen after double-strand break repair. Complete and precise excision of the repeat tract from normal and large expanded DMPK alleles in myoblasts from unaffected individuals, DM1 patients, and a DM1 mouse model could be achieved at high frequency by dual CRISPR/Cas9-cleavage at either side of the (CTG⋅CAG)n sequence. Importantly, removal of the repeat appeared to have no detrimental effects on the expression of genes in the DM1 locus. Moreover, myogenic capacity, nucleocytoplasmic distribution, and abnormal RNP-binding behavior of transcripts from the edited DMPK gene were normalized. Dual sgRNA-guided excision of the (CTG⋅CAG)n tract by CRISPR/Cas9 technology is applicable for developing isogenic cell lines for research and may provide new therapeutic opportunities for patients with DM1

miR-542 promotes mitochondrial dysfunction and SMAD activity and is raised in ICU Acquired Weakness

Rationale: Loss of skeletal muscle mass and function is a common consequence of critical illness and a range of chronic diseases but the mechanisms by which this occurs are unclear. Objectives: We aimed to identify miRNAs that were increased in the quadriceps of patients with muscle wasting and to determine the molecular pathways by which they contributed to muscle dysfunction. Methods: miR-542-3p/-5p were quantified in the quadriceps of patients with COPD and intensive care unit acquired weakness (ICUAW). The effect of miR-542-3p/5p was determined on mitochondrial function and TGF-β signaling in vitro and in vivo. Measurements and main results: miR-542-3p/5p were elevated in patients with COPD but more markedly in patients with ICUAW. In vitro, miR-542-3p suppressed the expression of the mitochondrial ribosomal protein MRPS10, and reduced 12S rRNA expression suggesting mitochondrial ribosomal stress. miR-542-5p increased nuclear phospho-SMAD2/3 and suppressed expression of SMAD7, SMURF1 and PPP2CA, proteins that inhibit or reduce SMAD2/3 phosphorylation suggesting that miR-542-5p increased TGF-β signaling. In mice, miR-542 over-expression caused muscle wasting, reduced mitochondrial function, 12S rRNA expression and SMAD7 expression, consistent with the effects of the miRNAs in vitro. Similarly, in patients with ICUAW, the expression of 12S rRNA and of the inhibitors of SMAD2/3 phosphorylation were reduced, indicative of mitochondrial ribosomal stress and increased TGF-β signaling. In patients undergoing aortic surgery, pre-operative levels of miR-542-3p/5p were positively correlated with muscle loss following surgery. Conclusion; Elevated miR-542-3p/5p may cause muscle atrophy in ICU patients through the promotion of mitochondrial dysfunction and activation of SMAD2/3 phosphorylation

Small heat-shock protein HSPB3 promotes myogenesis by regulating the lamin B receptor

One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR

Consolidation of an Olfactory Memory Trace in the Olfactory Bulb Is Required for Learning-Induced Survival of Adult-Born Neurons and Long-Term Memory

Background: It has recently been proposed that adult-born neurons in the olfactory bulb, whose survival is modulated by learning, support long-term olfactory memory. However, the mechanism used to select which adult-born neurons following learning will participate in the long-term retention of olfactory information is unknown. We addressed this question by investigating the effect of bulbar consolidation of olfactory learning on memory and neurogenesis. Methodology/Principal Findings: Initially, we used a behavioral ecological approach using adult mice to assess the impact of consolidation on neurogenesis. Using learning paradigms in which consolidation time was varied, we showed that a spaced (across days), but not a massed (within day), learning paradigm increased survival of adult-born neurons and allowed long-term retention of the task. Subsequently, we used a pharmacological approach to block consolidation in the olfactory bulb, consisting in intrabulbar infusion of the protein synthesis inhibitor anisomycin, and found impaired learning and no increase in neurogenesis, while basic olfactory processing and the basal rate of adult-born neuron survival remained unaffected. Taken together these data indicate that survival of adult-born neurons during learning depends on consolidation processes taking place in the olfactory bulb. Conclusion/Significance: We can thus propose a model in which consolidation processes in the olfactory bulb determine both survival of adult-born neurons and long-term olfactory memory. The finding that adult-born neuron survival durin

Cell-mediated exon skipping normalizes dystrophin expression and muscle function in a new mouse model of Duchenne Muscular Dystrophy

Cell therapy for muscular dystrophy has met with limited success, mainly due to the poor engraftment of donor cells, especially in fibrotic muscle at an advanced stage of the disease. We developed a cell-mediated exon skipping that exploits the multinucleated nature of myofibers to achieve cross-correction of resident, dystrophic nuclei by the U7 small nuclear RNA engineered to skip exon 51 of the dystrophin gene. We observed that co-culture of genetically corrected human DMD myogenic cells (but not of WT cells) with their dystrophic counterparts at a ratio of either 1:10 or 1:30 leads to dystrophin production at a level several folds higher than what predicted by simple dilution. This is due to diffusion of U7 snRNA to neighbouring dystrophic resident nuclei. When transplanted into NSG-mdx-Δ51mice carrying a mutation of exon 51, genetically corrected human myogenic cells produce dystrophin at much higher level than WT cells, well in the therapeutic range, and lead to force recovery even with an engraftment of only 3-5%. This level of dystrophin production is an important step towards clinical efficacy for cell therapy

Travelers With Cutaneous Leishmaniasis Cured Without Systemic Therapy

Guidelines recommend wound care and/or local therapy as first-line treatment for cutaneous leishmaniasis. An analysis of a referral treatment program in 135 travelers showed that this approach was feasible in 62% of patients, with positive outcome in 83% of evaluable patient

Progress in muscular dystrophy research with special emphasis on gene therapy

Duchenne muscular dystrophy (DMD) is an X-linked, progressive muscle-wasting disease caused by mutations in the DMD gene. Since the disease was described by physicians in the 19th century, information about the subject has been accumulated. One author (Sugita) was one of the coworkers who first reported that the serum creatine kinase (CK) level is elevated in progressive muscular dystrophy patients. Even 50 years after that first report, an elevated serum CK level is still the most useful marker in the diagnosis of DMD, a sensitive index of the state of skeletal muscle, and useful to evaluate therapeutic effects. In the latter half of this article, we describe recent progress in the therapy of DMD, with an emphasis on gene therapies, particularly exon skipping